The Antidepressant 5-HT2A Receptor Antagonists Pizotifen and Cyproheptadine Inhibit Serotonin-Enhanced Platelet Function

There is considerable interest in defining new agents or targets for antithrombotic purposes. The 5-HT2A receptor is a G-protein coupled receptor (GPCR) expressed on many cell types, and a known therapeutic target for many disease states. This serotonin receptor is also known to regulate platelet function. Thus, in our FDA-approved drug repurposing efforts, we investigated the antiplatelet activity of cyproheptadine and pizotifen, two antidepressant 5-HT2A Receptor antagonists. Our results revealed that cyproheptadine and pizotifen reversed serotonin-enhanced ADP-induced platelet aggregation in vitro and ex vivo. And the inhibitory effects of these two agents were found to be similar to that of EMD 281014, a 5-HT2A Receptor antagonist under development. In separate experiments, our studies revealed that these 5-HT2A receptor antagonists have the capacity to reduce serotonin-enhanced ADP-induced elevation in intracellular calcium levels and tyrosine phosphorylation. Using flow cytometry, we also observed that cyproheptadine, pizotifen, and EMD 281014 inhibited serotonin-enhanced ADP-induced phosphatidylserine (PS) exposure, P-selectin expression, and glycoprotein IIb-IIIa activation. Furthermore, using a carotid artery thrombosis model, these agents prolonged the time for thrombotic occlusion in mice in vivo. Finally, the tail-bleeding time was investigated to assess the effect of cyproheptadine and pizotifen on hemostasis. Our findings indicated prolonged bleeding time in both cyproheptadine- and pizotifen-treated mice. Notably, the increases in occlusion and bleeding times associated with these two agents were comparable to that of EMD 281014, and to clopidogrel, a commonly used antiplatelet drug, again, in a fashion comparable to clopidogrel and EMD 281014. Collectively, our data indicate that the antidepressant 5-HT2A antagonists, cyproheptadine and pizotifen do exert antiplatelet and thromboprotective effects, but similar to clopidogrel and EMD 281014, their use may interfere with normal hemostasis.


Introduction
Platelets are specialized anucleated cells that directly contribute to, and regulate hemostasis. Hemostasis is a physiological process that stops bleeding upon blood vessel injury. The inappropriate activation of hemostatic mechanisms and unrestrained platelet aggregation may lead to the development of thromboembolic events [1][2][3][4]. Therefore, it is critical to understand the mechanisms of platelet activation, in order to define novel pharmacological agents to reduce the adverse outcomes of unrestrained platelet activities.
Serotonin, or 5-hydroxytryptamine (5-HT), plays a key role in the development of arterial thrombosis [5]. It is predominantly synthesized/secreted into the blood stream by enterochromaffin cells in the gastrointestinal tract, and is rapidly taken up and stored in platelet dense granules [6]. As long as platelets do not aggregate, peripheral blood contains little or no free serotonin [7]. Upon platelet activation at the site of vessel injury, 5-HT is released from the dense granules in platelets. Serotonin or 5-HT by itself is a weak activator of platelet aggregation but it amplifies aggregation induced by other agonists including collagen, ADP, and epineph-rine [8,9]. Previous studies have shown a significant serotonindependent increase in platelet aggregation in depressed patients compared with controls, indicating serotonin plays an important role in the genesis of occlusive diseases [10,11].
G protein coupled receptors (GPCRs) in platelets have been extensively studied to identify targets for treating a multitude of cardiovascular events. One such receptor is the serotonin 5-HT 2A receptor, which belongs to the 5-HT 2 receptor family. The 5-HT 2A receptors are of significant clinical interest because they are involved in the mediation of mental disorders [12,13], and many cardiovascular diseases [14,15]. To this end, it has been previously shown that inhibition of serotonin 5-HT 2A receptor can improve coronary patency in in-vivo model of recurrent thrombosis [16]. Previous studies have also reported platelet aggregation is enhanced (e.g., hyperactive 5-HT 2A receptor signaling) in depressed patients [17][18][19][20][21]. This is because traditional therapies focus on elevating serotonin levels, but this approach can have serious side effects, including increased risk of serotonin syndrome and cardiovascular-related adverse events [22,23].
Despite the crucial role of serotonin and 5-HT 2A receptor activation in platelet function, there are currently no 5-HT 2A receptor antagonists approved by the Food and Drug Administration (FDA), for treatment of arterial thrombosis [24]. This is an important issue given the limitations of current antiplatelet therapies. Based on these considerations, we sought to investigate whether conventional FDA-approved antidepressant drugs, namely cyproheptadine and pizotifen, can be repurposed to ameliorate serotonin receptor-dependent platelet aggregation and thrombogenesis [25][26][27]. Our studies revealed that these drugs do have the capacity to inhibit serotonin-enhanced ADP-induced platelet aggregation in vitro, and ex vivo; similar to EMD 281014, another potent and selective 5-HT 2A receptor antagonist [28][29][30]. These drugs also have the capacity to inhibit serotonin-enhanced ADPinduced elevation in intracellular calcium and tyrosine phosphorylation. We also observed that serotonin-enhanced ADP-stimulated platelet phosphatidylserine (PS) exposure, P-selectin expression, and glycoprotein (GP) IIb-IIIa activation were inhibited by cyproheptadine, pizotifen and EMD 281014, in vitro. Moreover, cyproheptadine and pizotifen were found to significantly prolong occlusion time in mouse thrombosis model, but normal hemostasis may also be interfered, as demonstrated in tail bleeding time experiments. Notably, the in vivo activities of cyproheptadine and pizotifen were determined to be comparable to that of the clinically-relevant and commonly prescribed antithrombotic drug, clopidogrel.

Cyproheptadine and Pizotifen Inhibit Serotoninenhanced ADP-induced Human Platelet Aggregation in vitro
As a known weak activator of platelet aggregation [8,31], serotonin did not induce platelet aggregation in human PRP (Fig. 1A). On the other hand, weak and reversible platelet aggregation was observed when platelets were stimulated with submaximal concentration of ADP (i.e., 1 mM). Simultaneous addition of serotonin (15 mM) resulted in significant potentiation of platelet aggregation induced by 1 mM of ADP (Fig. 1A), demonstrating that serotonin has the ability to enhance ADPinduced platelet aggregation. Next, we investigated whether the antidepressant 5-HT 2A receptor antagonists, namely cyproheptadine and pizotifen, can be repurposed as antiplatelet agents, and used to inhibit serotonin-enhanced ADP-induced platelet aggregation in vitro. Aggregation studies indicated that cyproheptadine (0.1-10 nM) and pizotifen (0.01-1 nM) have the capacity to dosedependently inhibit serotonin-enhanced ADP-induced platelet aggregation ( Fig. 1B-1C). The first set of control experiments was performed using EMD 281014, a potent and selective 5-HT 2A receptor antagonist; its antiplatelet activity has yet to be determined. Our result indicated that EMD 281014 (10-40 nM) also dose-dependently inhibited human platelet aggregation in vitro (Fig. 1D). To verify that cyproheptadine and pizotifen specifically antagonize serotonin-enhanced platelet function, and that they do not affect platelet activity in the absence of serotonin, a second series of experiments was performed. As expected, cyproheptadine (10 nM) pizotifen (1 nM), and EMD 281014 (40 nM) were found to inhibit (15 mM) serotonin-induced limited platelet activation (i.e., shape change; Fig. 1E), but neither agent (with the exception of EMD 281014) exerted any effects on ADP-induced platelet aggregation (Fig. 1F), or on non-stimulated resting platelets (Fig. 1G).

Cyproheptadine and Pizotifen Inhibit Serotoninenhanced U46619-induced Human Platelet Aggregation in vitro
Given that serotonin is a weak agonist that amplifies platelet aggregation responses, we investigated its effects on aggregation induced by another agonist, namely U46619 which is an agonist for the thromboxane receptor. Using U46619, a potent agonist of platelet aggregation, we were able to verify that serotonin has the ability to enhance platelet aggregation induced by submaximal concentration, i.e., 0.125 mM of U46619 ( Fig. 2A). Again, separate aggregation studies revealed that cyproheptadine (0.1-250 nM) and pizotifen (0.1-30 nM) have the capacity to dosedependently inhibit serotonin-enhanced U46619-induced platelet aggregation, in vitro ( Fig. 2B-2C). EMD 281014 (5-20 nM) also has the capacity to dose-dependently inhibit serotonin-enhanced U46619-induced platelet aggregation (Fig. 2D). It was further demonstrated that each of the 5-HT 2A receptor antagonist used did not exert any effect on U46619-induced platelet aggregation, with the exception of EMD 281014 (Fig. 2E); this is consistent with what was observed with ADP ( Fig. 1E-1G), and further supports that cyproheptadine and pizotifen do specifically inhibit serotonin-enhanced platelet function induced by multiple agonists.

Cyproheptadine and Pizotifen Inhibit Serotoninenhanced ADP-induced Elevation in Intracellular Calcium in Human Platelets in vitro
In additional control experiments, the capacity of these agents to inhibit the elevation in intercellular calcium that is serotoninenhanced ADP-induced was also assessed. Our measurements revealed that cyproheptadine (10 nM), pizotifen (1 nM), and EMD 281014 (40 nM) all significantly reduced platelet intracellular calcium levels that are triggered by 1 mM ADP in the presence of 15 mM serotonin (Fig. 5A). In another set of control experiments, western blot analysis was performed in order to assess the ability of cyproheptadine and pizotifen to inhibit serotonin-enhanced ADP-triggered tyrosine phosphorylation of Src family kinases. Analysis indicated that 5-HT 2A receptor antagonists, i.e., cyproheptadine (10 nM), pizotifen (1 nM), and EMD 281014 (40 nM) all have the ability to inhibit 15 mM serotonin-enhanced ADP-induced (1 mM) tyrosine phosphorylation in platelets, in vitro (Fig. 5B).

Cyproheptadine and Pizotifen Inhibit Serotoninenhanced ADP-induced Mouse Platelet Aggregation ex vivo
In order to investigate if the antiplatelet effects of cyproheptadine and pizotifen can be manifested under chronic dosing conditions in live animals, ex vivo mouse aggregation experiments were first performed. Using platelets isolated from mice injected with pharmacologically-relevant doses of 5-HT 2A receptor antagonists, once daily, for 5 days, our results demonstrated that, compared to the vehicle control (Fig. 6A), both cyproheptadine (1 mg/kg, IP) and pizotifen (3 mg/kg, IP) almost completely inhibited serotonin-enhanced ADP-induced platelet aggregation (Fig. 6B, and 6C). Similarly, chronic dosing with EMD 281014 (5 mg/kg, IP), inhibited serotonin-enhanced ADP-induced platelet aggregation (Fig. 6D), and (interestingly) exerted inhibitory effects on ADP-induced platelet aggregation, in the absence of serotonin (Fig. 6D). Together, our findings indicate that cyproheptadine and pizotifen's antiplatelet effects are sustained following a chronic dosing regimen. It is noteworthy that the aforementioned doses and literature [29,30,[32][33][34][35][36][37][38] guided our doses selection for the in vivo experiments, i.e., pharmacologically relevant doses.
Cyproheptadine and Pizotifen Inhibit Serotoninenhanced ADP-induced Phosphatidylserine Exposure, Pselectin Expression, and Glycoprotein IIb-IIIa Activation, in Human Platelets Next, we assessed whether the aforementioned 5-HT 2A receptor antagonists exert inhibitory effects on separate platelet functional responses, i.e., PS exposure, P-selectin expression, and GPIIb-IIIa activation, which are known to also be enhanced by serotonin. In platelets stimulated with submaximal concentration of ADP (1 mM) alone or ADP with serotonin (15 mM), an increase in mean fluorescence intensity (MFI), indicative of PS exposure, Pselectin expression, and GPIIb-IIIa activation, was observed ( Fig. 7A-G). Moreover, in platelets pre-incubated with cyproheptadine (10 nM), and pizotifen (1 nM), resulted in a dramatic reversal of PS exposure, P-selectin expression, and GPIIb-IIIa activation that are ADP-stimulated serotonin potentiated, as follows: 1.

Cyproheptadine and Pizotifen Prolong Occlusion Time in a Carotid Artery Injury-induced Thrombosis Model in Mice
To evaluate the potential of cyproheptadine and pizotifen to alleviate thrombotic events in vivo, a mouse carotid artery FeCl 3 injury model was used. Our studies revealed that mice treated with 1 mg/kg of cyproheptadine exhibited significantly increased time to occlusion as compared to vehicle-treated animals (787.4684.08 sec versus 375.3631.89 sec; mean, p,0.0001;  thrombus formation, and may be used to protect against arterial thrombosis. In addition, mice treated with 5 mg/kg of EMD 281014 also exhibited significant increase time to occlusion as compared to vehicle-treated mice (879.96270.0 sec versus 375.3631.89 sec; mean, p,0.0431), which does not significantly differ from that for cyproheptadine-or pizptifen-treated mice. These data suggest that the antidepressant 5-HT 2A receptor antagonists, cyproheptadine and pizotifen, as well as EMD 281014 exert thromboprotective properties in vivo.
To provide a clinically relevant standard for comparing the effects of cyproheptadine and pizotifen on thromboprotection, the most widely prescribed antiplatelet agent clopidogrel was chosen. Our result revealed that time to vessel occlusion in mice treated with 6 mg/kg of clopidogrel is significantly prolonged compare to controls (987.66196.5 sec versus 375.3631.89 sec; mean, p,0.0022), but is comparable to those treated with 5-HT 2A receptor antagonists. This suggests that the antidepressant 5-HT 2A receptor antagonists are, perhaps, just as effective as the standard treatment of clopidogrel, at improving vessel occlusion time during a thrombotic event.

Cyproheptadine and Pizotifen Prolong Tail Bleeding Time in Mice
We next examined the effect of cyproheptadine, pizotifen and EMD 281014 on hemostasis. Compared to vehicle-injected animals, mice injected with the 5-HT 2A receptor antagonists exhibited significant increase in tail bleeding times (vehicle = 213.1681.03 sec; mean, CYP = 643.1697.78 sec; mean, p,0.0038, Pizo = 714.4696.78 sec; mean, p,0.0011; EMD 281014 = 558.9667.12 sec; mean, p,0.0047; Fig. 8B). The prolongation of tail bleeding correlates to increased risk of impaired hemostasis, and this risk of bleeding is comparable to that associated with clopidogrel (793.6658.17 sec; mean).

Discussion
Cyproheptadine and pizotifen are two FDA-approved antidepressants that target and antagonize serotonin 5-HT 2A receptors in CNS, and have been commonly used to treat depressive disorders [27,37]. In the periphery, 5-HT 2A receptors are located in vascular smooth muscles and platelets; and they have been implicated in regulatory processes including vasoconstriction and platelet aggregation [8,15]. To this end, several potent and selective antagonists of the 5-HT 2A receptor were developed and tested and have demonstrated antiplatelet and antithrombotic activities [29,[39][40][41][42][43][44]. Unfortunately however, none of these molecules has made it into clinical practice, at least in the context of thrombogenesis [5,9,24]. Consequently, we decided to investigate whether the aforementioned antidepressant 5-HT 2A receptor antagonists may be repurposed for antiplatelet therapy [25,26,41].
Our initial in vitro aggregometry characterization revealed that cyproheptadine and pizotifen, have the capacity to dose-depen- dently inhibit serotonin-enhanced ADP-induced aggregation, but are less potent on mouse compared to human platelets. This is perhaps because the human and mouse 5-HT 2A receptor sequences, including their ligand binding domain(s), while highly conserved, are not identical, [45]. Nonetheless, the concentrations of either 5-HT 2A receptor antagonist needed to inhibit platelet aggregation in both human and murine platelets are in nM ranges, demonstrated a superior pharmacological profile.
The capacity of cyproheptadine, pizotifen and EMD 281014 to reverse the serotonin enhanced platelet function was further assessed using intracellular calcium measurements, and the activation of Src family kinases. It was found that these agents, at concentrations sufficient to almost completely inhibit serotoninenhanced ADP-induced aggregation, also reduced intracellular calcium and Src activation. In separate control studies, the aforementioned 5-HT 2A receptor antagonists were also found to inhibit a host of platelet functional responses that are serotoninenhanced ADP-induced, i.e., PS exposure, P-selectin expression, and GPIIb-IIIa activation. These findings indicated that cyproheptadine, pizotifen and EMD 281014 can down-regulate signal transduction associated with serotonin-enhanced platelet activation, and are consistent with the aggregation data. It appears that the magnitude of inhibition of GPIIb-IIIa activation by 5-HT 2A receptor antagonists does not correspond to their ability to inhibit aggregation, which we believe is due to experimental variability.
Given that patients are conventionally prescribed antidepressants or antiplatelet drugs for an extended period of time, we next sought to examine if the antiplatelet effects of these antidepressants can also be manifested in murine platelets under chronic dosing conditions. Thus, ex vivo experiments were performed following intraperitoneal (IP) injections of cyproheptadine (1 mg/kg) or pizotifen (3 mg/kg) once daily for 5 days. It is noteworthy that these doses were derived from the literature because of their pharmacological relevance for depressive disorders [29,30,[32][33][34][35][36], to determine whether the same dose is capable of exerting dual pharmacological activity. Our data indicated that repeated injections with cyproheptadine and pizotifen did reverse serotonin-enhanced aggregation triggered by low-dose ADP.
To further determine if cyproheptadine and pizotifen's antiplatelet properties can be translated to live animals, we performed mouse carotid artery thrombosis experiments. It was observed that the time to vessel occlusion drastically increased in mice treated with cyproheptadine or pizotifen. This finding indicates that these 5-HT 2A receptor antagonists have the ability to block serotonin-enhanced thrombogenesis, which derives from their ability to interfere with platelet activation. Clopidogrel was employed as a positive control, to provide a clinical benchmark for comparing the efficacy of cyproheptadine and pizotifen as alternative thromboprotective agents. Analysis revealed that while clopidogrel treatment did significantly prolonged occlusion times, its magnitude did not significantly differ from that observed in cyproheptadine-, and pizotifen-treated mice. Given their different target receptors (clopidogrel on P2Y 12 receptors) these 5-HT 2A receptor antagonist may be able to serve as alternative medications, or complement to clopidogrel, given that dual therapy with clopidogrel is common clinical practice for thromboprotection [9,46,47].
In addition, to provide a relevant control for antidepressant 5-HT 2A receptor antagonists cyproheptadine and pizotifen, EMD 281014 was chosen for comparison. Analysis revealed that while EMD 281014 also has the capacity to prolong occlusion times, large biological variations were observed. Given that EMD 281014, cyproheptadine and pizotifen are all 5-HT 2A receptor antagonists, but lower doses of cyproheptadie and pizotifen were needed to achieve higher significant prolongation of occlusion times and more consistently with less variation, this suggests, cyproheptadine and pizotifen may be more predictable and effective antiplatelet agents compared to EMD 281014.
Increased risk of bleeding is a common concern for patients on any antiplatelet agent, and thus it was investigated using cyproheptadine and pizotifen. Our results indicated that the tail bleeding times were significantly increased in animals treated with either cyproheptadine or pizotifen; similar to what was observed in mice treated with clopidogrel and EMD 281014. These data indicate that continuous treatment with cyproheptadine or pizotifen may result in increased bleeding risk, similar to the (currently) most commonly-prescribed antiplatelet drug clopidogrel. Given that cyproheptadine and pizotifen's antithrombotic effects are comparable to clopidogrel, we believe that depressive patients with primed platelet activity or high risk of thrombosis will likely benefit from one of these agents. Repurposing old drugs for new applications can be advantageous as this approach could dramatically reduce the cost and time required for drugs to be approved for market and used in clinics. Furthermore, if modeled over their chemistry, structural analogs or derivatives of these 5-HT 2A receptor antagonists may be developed, with much more competitive pharmacological profiles. Lastly, while our data may argue against it, we cannot exclude contribution from vasculature or other 5-HT 2A receptors to the observed antiplatelet phenotype.
In summary, our studies demonstrate that FDA-approved antidepressants, 5-HT 2A receptor antagonists pizotifen and cyproheptadine (at standard dosages), exert antiplatelet activity, and thus can be repurposed for the treatment of thromboembolic disorders; albeit their use may interfere with normal hemostasis.

Materials and Methods
The human donor blood part of the work has been approved by the Institutional Review Board at Western University of Health Sciences, and donors were asked to sign a written consent, and a subjects' bill of rights. The animal work part of these studies has been approved by Institutional Animal Care and Use Committee at Western University of Health Sciences.

Reagents and Materials
Serotonin hydrochloride, pizotifen and ADP were obtained from Sigma Aldrich (St. Louis, MO), cyproheptadine and EMD 281014 were obtained from Tocris Bioscience (Bristol, UK), clopidogrel was purchased from LKT Laboratories, Inc. (St. Paul, MN), stir bars and other disposables were from Chrono-Log (Havertown, PA), and U46619 was obtained from Cayman Chemical (Ann Arbor, MI). Src antibody, FITC-conjugated Annexin V, anti-P-selectin, and PAC-1 antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA). The antiphosphotyrosine antibody was from BD Biosciences, (Franklin Lakes, NJ). Fura-2 acetoxymethyl ester (fura-2/AM) and Plur-onicH F-127 were from Invitrogen (Grand Island, NY). The C57BL/6 mice were obtained from Jackson laboratory (Bar Harbor, ME). Platelet count was determined using an automated hematology analyzer (Drew Scientific Dallas, TX). Animals C57BL/6 mice were obtained from Jackson Laboratory (Bar Harbor, ME). Mice were housed in groups of 1-4 at 24uC, under 12/12 light/dark cycles, with access to water and food ad libitum. All experiments involving animals were perform in compliance with the institutional guidelines, and were approved by the Western University of Health Sciences Institutional Animal Care and Use Committee.

Human and Murine Platelet Preparation
Blood was drawn from healthy volunteers who denied taking any medication for 1 week prior to collection, or from C57BL/6 mice (8-10 weeks old). Mice were anesthetized and blood was collected from the heart. Coagulation was inhibited by 3.8% w/v sodium citrate solution (1 part sodium citrate to 9 parts blood).
Human or mouse platelet rich plasma (PRP) was obtained by centrifugation at room temperature. Platelets were counted with automated hematology analyzer and their count adjusted to 7610 7 platelets/ml, prior to each experiment.

Ex vivo Platelet Aggregation
Mice were injected with vehicle (DMSO), or pharmacologically/therapeutically relevant doses [29,30,[32][33][34][35][36][37][38] of cyproheptadine (1 mg/kg), pizotifen (3 mg/kg), clopidogrel (6 mg/kg), or EMD 281014 (5 mg/kg) using the intraperitoneal (IP) route once daily for 5 days; in an attempt to mimic chronic administration of these drugs in patients. Mice were sacrificed two hours post last injection, and their blood collected. Platelets (with counts adjusted as described before) were stimulated with 1 mM ADP in the presence or absence of 15 mM serotonin, and platelet aggregation was measured. Each experiment was repeated at least 3 times, with blood pooled from at least eight mice each time.

Intracellular Calcium Measurement in Platelets
Intra-platelet calcium was measured using Fura-2-acetoxymethyl ester (Fura-2AM) as described [48]. Mouse platelets (2.0610 8 /mL) were labeled with 12.5 mM Fura-2AM and 0.2% Pluronic F-127 in HEPES/Tyrode buffer (pH 7.4) for 45 min at 37uC. After washing, the platelets were resuspended without apyrase to a concentration of 2.0610 8 /mL. Samples (1 mL) were added to siliconized cuvettes, recalcified with 0.7 mM CaCl 2 , and incubated in the presence or absence of cyproheptadine (10 nM), pizotifen (1 nM) or EMD 281014 (40 nM) for 5 minutes and then stimulated with ADP (1 mM), serotonin (15 mM) or ADP and serotonin together for 3 minutes with constant stirring. Fluorescence was analyzed by excitation at 340 nm and 380 nm and emission was measured at 509 nm using a model LS50B Luminescence Spectrometer (Perkin-Elmer Instruments, Shelton, CT). The ratio of fura-2 emissions was calculated simultaneously using FL WinLab software and converted to [Ca 2+ ] i , as described previously [49].

Immunoprecipitation and Immunoblotting
Immunoprecipitation was carried out as described in Karim et al [48]. Briefly, human platelets were incubated in the presence or absence of Cyproheptadine (10 nM), pizotifen (1 nM) or EMD 281014 (40 nM) for 5 minutes and then stimulated with ADP (1 mM), serotonin (15 mM) or ADP and serotonin together for 3 minutes followed by lysis with 26 lysis buffer (40 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM Na 2 EDTA, 2 mM EGTA, 2% Triton X-100, 2% sodium deoxycholate, 5 mM sodium pyrophosphate, 2 mM Na 3 VO 4 , and protease inhibitor cocktail). Platelet lysates were clarified by centrifugation and the supernatants were precleared by incubating with rabbit IgG and then incubated with anti-Src antibody. Immunoprecipitates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P PVDF membranes (Bio-Rad, Hercules, CA). They were then probed with the primary antibodies (Src and anti-phosphotyrosine) and visualized with horseradish peroxidase-labeled anti-rabbit IgG or anti-mouse IgG as required. The antibody binding was detected using enhanced chemiluminescence substrate (Thermo Scientific, Rockford, IL). Images were obtained with ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA) and quantified with Image Lab software Version 4.1 (Bio-Rad, Hercules, CA).

Flow Cytometric Analysis
Human platelets (2610 8 ) were incubated in the presence or absence of Cyproheptadine (10 nM), pizotifen (1 nM) or EMD 281014 (40 nM) for 5 minutes and then stimulated with ADP (1 mM), serotonin (15 mM) or ADP and serotonin together for 3 minutes. The reactions were stopped by fixing the platelets with 2% formaldehyde for 30 min at room temperature. Finally, platelets were incubated with FITC-conjugated Annexin V, anti-P-selectin, or PAC-1 antibodies at room temperature for 30 min in the dark. Finally, the platelets were diluted 2.5-fold with HEPES/ Tyrode buffer (pH 7.4). The samples were transferred to FACStubes and fluorescent intensities were measured using a BD Accuri C6 flow cytometer and analyzed using CFlow Plus (BD Biosciences, Franklin Lakes, NJ).

In vivo Thrombosis Model
These studies were performed as described previously [50]. Briefly, mice 8-10 weeks old received IP injections of cyproheptadine, pizotifen, clopidogrel, or EMD 281014 at doses described before, and were anesthetized with isoflurane. Then, the left carotid artery was exposed and cleaned, and baseline carotid artery blood flow was measured with Transonic micro-flowprobe (0.5 mm, Transonic Systems Inc., Ithaca, NY). After stabilization of blood flow, 7.5% ferric chloride (FeCl 3 ) was applied to a filter paper disc (1-mm diameter) that was immediately placed on top of the artery for 3 min. Blood flow was continuously monitored for 30 min, or until blood flow reached stable occlusion (zero blood flow for 2 min). Data was recorded and time to vessel occlusion was calculated as the difference in time between stable occlusion and removal of the filter paper (with FeCl 3 ). An occlusion time of 30 min was considered as the cut-off time for the purpose of statistical analysis.

Tail Bleeding Time
Mice were IP injected with cyproheptadine, pizotifen, clopidogrel, EMD 281014 or vehicle once daily for 5 days, as described before. Hemostasis was examined using the tail transection technique [50]. Briefly, mice were anesthetized and placed on a 37uC homeothermic blanket. Tail was transected 5 mm from the tip using a sterile scalpel. After transection, the tail was immediately immersed in saline (37uC, constant temperature) and the time to bleeding cessation was measured. Bleeding time of 15 min was considered as the cut-off time for the purpose of statistical analysis.

Statistical Analysis
All experiments were performed at least three times. Analysis of the data was performed using GraphPad PRISM statistical software (San Diego, CA) and presented as mean 6 SEM. The Mann-Whitney test was used for the evaluation of differences in mean occlusion and bleeding times. Analysis was also conducted using t-test, and similar results were obtained. Significance was accepted at P,0.05 (two-tailed P value), unless stated otherwise.